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The Nonclassical Major Histocompatibility Complex Molecule Qa-2 Protects Tumor Cells from NK Cell- and Lymphokine-Activated Killer Cell-Mediated Cytolysis¹

Center for Immunology, Departments of Microbiology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390

	Abstract

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The cytotoxic activity of NK cells is regulated by class I MHC proteins. Although much has been learned about NK recognition of class I autologous targets, the mechanisms of NK self-tolerance are poorly understood. To examine the role of a nonpolymorphic, ubiquitously expressed class Ib Ag, Q9, we expressed it on class I-deficient and NK-sensitive B78H1 melanoma. Presence of this Qa-2 family member on tumor cells partially protected targets from lysis by bulk lymphokine-activated killer (LAK) cells. H-2K^b-expressing B78H1 targets also reduced LAK cell activity, while H-2D^b offered no protection. Importantly, blocking with F(ab')₂ specific for Q9 or removal of this GPI-attached molecule by phospholipase C cleavage restored killing to the level of vector-transfected cells. Experiments with LAK cells derived from H2^b SCID and B6 mice established that NK1.1⁺TCR^- NK and NK1.1⁺TCR⁺ LAK cells were the prevalent cytolytic populations inhibitable by Q9. Treatment of mice with poly(I:C) also resulted in generation of Q9-regulated splenic cytotoxicity. LAK cells from different mouse strains responded to Q9, suggesting that the protective effect of this molecule is not detectably influenced by Ly49 polymorphisms or the presence/absence of Q9 in NK-harboring hosts. We propose that Q9 expressed on melanoma cells serves as a ligand for yet unidentified NK inhibitory receptor(s) expressed on NK1.1⁺ NK/T cells.

	Introduction

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Recent studies of the class Ia/class Ib family of MHC Ags suggest that these molecules carry out diverse functions in the adaptive and innate immune systems. In addition to presenting pathogen- and tumor-derived ligands to thymically educated T cells, they participate in regulation of NK cell cytotoxicity and in little understood interactions with T cells, intestinal intraepithelial lymphocytes, macrophages, and others. In the mouse system, three categories of class I molecules were reported to interact with NK1.1⁺ cells: class Ia MHC H-2K and H-2D (1, 2, 3), class Ib MHC Qa-1 Ag (4, 5, 6), and non-MHC class Ib protein CD1d (7). Each of these molecules plays a different role in NK cell biology and employs a different set of NK receptors to inhibit or activate their cytotoxicity and/or modulate cytokine production. In H2^b B6 mice, 25% of NK cells are devoid of the known H2^b class I-specific inhibitory receptors, suggesting that their immunological tolerance toward self is regulated by a set of novel, yet undiscovered NK/target interactions (8).

We have addressed a possibility that the Q region-encoded nonpolymorphic class Ib MHC molecule Q9^b (9) is involved in NK cell regulation. The Q9 Ag is a member of the Qa-2 family (10). Its primary amino acid sequence is as similar to class Ia as the alleles of H-2K and H-2D are to each other (9). The three-dimensional crystallographic structure of Q9 is highly homologous to class Ia structures as well as to the class Ib protein HLA-E (11). Furthermore, Q9 binds a wide repertoire of TAP-dependent self and nonself nonameric peptides (12), suggesting that Q9 can alert the immune system to the presence of intracellular pathogen infections or malignant transformations. Despite its Ag-presenting properties and the ability to serve as an allogeneic CTL target (13), Q9 is not known to function as a restricting element for pathogen-specific CTL (14, 15), a feature that has been attributed to the inability of Q9 to promote efficient positive selection of thymic T cells (16). The Q9 Ags have a wide tissue distribution and are expressed in immunologically privileged sites/organs/cells: anterior chamber of the eye (17), hair follicles (18), embryo and placenta (19), oocytes and blastocysts (20), and sperm in testis (21). We report in this study that Q9 expressed on melanocyte-derived tumor cells inhibits cytotoxic activity of lymphokine-activated killer (LAK)³ cells. Accordingly, we propose that this molecule plays a role in innate immunity and/or in protecting syngeneic cells from NK-mediated killing, particularly in immunologically privileged tissues, where expression of class Ia and Qa-1 complexed with class Ia leader peptides is very low.

	Materials and Methods

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Mice

C57BL/6J (abbreviated B6, H2^b, Qa-2⁺), B6.K1 (H2^b, Qa-2^-), B6 SCID (SCID, H2^b, Qa-2⁺), BALB/cJ (H2^d, Qa-2⁺), 129/J (H2^b, Qa-2⁺), C3H/HeJ (C3H, H2^k, Qa-2^-), ₂-microglobulin (₂m) knockout (₂m^-/-, H2^b), TAP^-/- (H2^b), and K^b-D^b- (H2^b) mice were either bred and maintained in the Department of Microbiology animal colony at University of Texas Southwestern Medical Center (Dallas, TX) or purchased from The Jackson Laboratory (Bar Harbor, ME). Adult mice (older than 8 wk) were used for all experiments.

Cell lines and cell culture

B78H1 (22) and GM-CSF-transduced B78H1 (23) melanoma cells were generously provided by Drs. H. I. Levitsky (Johns Hopkins School of Medicine, Baltimore, MD) and S. Ostrand-Rosenberg (University of Maryland Baltimore County, Baltimore, MD). Cells were cultured in 50% DMEM/50% RPMI 1640 supplemented with 10% FBS, 1 mM sodium pyruvate (Life Technologies, Grand Island, NY), 0.1 mM nonessential amino acids (Life Technologies), and 10 U/ml penicillin/10 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO). NK-sensitive YAC-1 lymphoma cells and NK-resistant P815 mastocytoma cells were provided by Dr. M. Bennett (University of Texas Southwestern, Dallas, TX) and were cultured in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FBS, 50 µM 2-ME (Sigma-Aldrich), 0.1 mM nonessential amino acids, 50x diluted essential amino acids (Life Technologies), 1 mM sodium pyruvate, 10 mM HEPES (Life Technologies), and penicillin/streptomycin). All cell lines were grown at 37°C and 5% CO₂. For IFN- induction, cells were treated with 20 U/ml recombinant mouse IFN- (Sigma-Aldrich) for 3 days.

Antibodies

The following anti-class I MHC Abs were used in this study: anti-Qa-2 mAb 1-1-2 (BD PharMingen, San Diego, CA), anti-H-2K^b mAb Y3 (24), anti-Qa-2/K^b mAb 20-8-4 (25), anti-H-2D^b mAb 28-14-8 (25), and anti-₂m mAb S19.8 (26) with FITC-goat anti-mouse IgG (Cappell, Durham, NC) used as a secondary Ab; biotinylated (bio-) anti-Qa-2 mAb M46, bio-Y3, and bio-S19.8 followed by PE-conjugated streptavidin (BioSource International, Camarillo, CA). The following mAbs purchased from BD PharMingen were also used: Fc block anti-mouse CD16/CD32 2.4G2 mAb, PE-conjugated anti-NK1.1 PK136 mAb, allophycocyanin- and FITC-conjugated anti-TCR -chain H57-597 mAb, allophycocyanin-conjugated anti-CD3 chain 145-2C11 mAb, PerCP-conjugated anti-CD8 53-6.7, FITC-conjugated anti-CD4 H129.19 mAb, FITC-conjugated anti-CD8.2 53-5.8 mAb, FITC-conjugated anti-CD19 1D3 mAb, FITC-conjugated anti-Ly49C 5E6 mAb, and anti-CD94 18d3 mAb. Isotype standards were also purchased from BD PharMingen: bio-, PE-, and FITC-conjugated mouse IgG2a, G155-178, allophycocyanin-conjugated hamster IgG, group 2 Ha4/8, allophycocyanin-conjugated hamster IgG, group 1 A19-3, PerCP- and FITC-conjugated rat IgG2a R35-95, and FITC-conjugated rat IgG1 R3-34. Blocking Ab 20-8-4 F(ab')₂ were generated by Dr. M. Bennett. Anti-Ly49C/I 5E6 and anti-Ly49G2 4D11 F(ab')₂ were generous gifts from Dr. M. Bennett. Control IgG F(ab')₂ were purchased from Caltag Laboratories (Burlingame, CA). Murine NKG2D-Ig fusion protein, control human Ig fusion protein, and anti-human Ig PE were generously provided by Dr. L. L. Lanier (University of California, San Francisco, CA) and used as previously described (27).

Transfection and selection

B78H1 cells expressing cell surface Q9 (designated Q9.A7) were derived by cotransfection with Q9-expressing plasmid Q9/pcDNA1 (28) and plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA), which carries the neomycin resistance gene. The Q9-expressing clone Q9.A7 was subsequently supertransfected with TAP2 full-length cDNA in pcDNA1neo (TAP2/pcDNA1neo; generously provided by Dr. J. J. Monaco, University of Cincinnati, Cincinnati, OH) and plasmid pcDNA3.1Hygro (Invitrogen), which carries the hygromycin resistance gene to generate Q9⁺TAP⁺ clone Q9TAP.C1. Q9⁺TAP⁺ clones Q9TAP.11 and Q9TAP.17 were generated by cotransfecting B78H1 and GM-CSF-transduced B78H1, respectively, with Q9/pcDNA1 and TAP2/pcDNA1neo. K^b+TAP⁺ clones K^bTAP.1-2.9 and K^bTAP.1-2.25 were generated by cotransfecting B78H1 with K^b/pcDNA3.1 and TAP2/pcDNA1neo. D^b+TAP⁺ clones D^bTAP.1-3.4 and D^bTAP.1-3.5 were generated by cotransfecting B78H1 with D^b/pcDNA1 and TAP2/pcDNA1neo. Control B78H1 transfectants were derived by transfection with either pcDNA3.1 plasmid alone (vector) or TAP2/pcDNA1neo alone (TAP2). Transfections were performed using the FuGENE 6 transfection reagent according to the manufacturer’s recommendations (Boehringer Mannheim, Indianapolis, IN). Briefly, 1 x 10⁵ cells were plated in a six-well tissue culture plate and cultured overnight. One microgram of each plasmid was used, except when cotransfections with antibiotic resistance gene-carrying plasmids were performed, in which case only 0.1 µg of antibiotic resistance gene-carrying plasmid was used. Cells were exposed to the transfection mixture overnight, washed, then grown in tissue culture medium for 2 days. Selection of Q9-expressing B78H1 cells was performed using tissue culture medium supplemented with 800 µg/ml G418 (Life Technologies), and clones were generated by limiting dilution and screened for Q9 expression by flow cytometry. The Q9-positive clone Q9.A7 was maintained in medium supplemented with 400 µg/ml G418. Selection and maintenance of TAP2-expressing Q9TAP.C1 cells was performed using medium additionally supplemented with 200 µg/ml hygromycin (Invitrogen). Clones were generated by limiting dilution and TAP2 expression was confirmed by RT-PCR. Q9TAP.11, Q9TAP.17, K^bTAP.1-2.9, K^bTAP.1-2.25, D^bTAP.1-3.4, and D^bTAP.1-3.5 were generated by single cell sort of bulk transfections. All clones were monitored by flow cytometry and RT-PCR for presence of class I and TAP2 expression and maintained in medium supplemented with 400 µg/ml G418.

Flow cytometry

For target cell staining, 1 x 10⁶ cells were washed once in staining buffer (PBS with 1% FCS and 0.1% sodium azide) and pelleted in a 1-ml polystyrene conical tube. A saturating amount of primary Ab was added to the cell pellet in a volume of 100 µl, vortexed, and incubated on ice for 15 min. Excess unbound Ab was removed by washing the suspension once with staining buffer. A dilution of FITC-labeled secondary Ab was added in a final volume of 100 µl and incubated on ice for 15 min. The samples were washed twice, resuspended in 300 µl of staining buffer, and filtered through 35-µm nylon mesh. A total of 1 x 10⁴ cells were collected on a FACScan flow cytometer (BD Biosciences, Palo Alto, CA). For effector cell staining, 1 x 10⁶ cells were washed once in staining buffer then pelleted in 96-well U-bottom polystyrene plates. FcR were blocked by incubating cells with 1 µg of anti-CD16/CD32 mAb in 20 µl of staining buffer for 15 min on ice. Effector cells were either analyzed for NK1.1 expression using single-color flow cytometry by staining with PE-conjugated anti-NK1.1 mAb or a detailed analysis was performed using multicolor flow cytometry. For multicolor analysis, a saturating amount of experimental Ab(s) was added to samples in a volume of 100 µl and samples were incubated on ice for an additional 30 min. The stained cells were washed twice, resuspended in 300 µl of staining buffer, and filtered through 35-µm nylon mesh. A total of 1 x 10⁴ cells were collected on a FACSCalibur flow cytometer (BD Biosciences). Fluorescence compensation was performed when the samples were analyzed by multicolor flow cytometric analysis. Gates were set using forward and side scatter parameters to exclude dead cells. All data was analyzed using CellQuest version 3.1f software (BD Biosciences).

RNA isolation, cDNA synthesis, and RT-PCR

Total RNA was isolated using the RNA STAT-60 method (Tel-Test, Friendswood, TX) as previously described (17). First-strand cDNA was synthesized from 5 µg of RNA in a reaction volume of 20 µl using the Life Technologies SuperScript II synthesis kit according to the manufacturer’s instructions. PCR was performed using the HotStarTaq DNA polymerase system (Qiagen, Valencia, CA). A total of 0.5–2 µl of cDNA was added to a total PCR mixture of 25 µl containing 2.5 µl of PCR buffer containing Tris-Cl, KCl, (NH₄)₂SO₄, and MgCl₂, 5 µl of Q-Solution, 0.5 µl of 10 mM dNTPs, 1 µl of 20 µM upstream primer, 1 µl of 20 µM downstream primer, 0.625 U of HotStarTaq DNA polymerase, and diethylpyrocarbonate-treated sterile H₂O. The primer pair TAP2F.1 (5'-GATCAACCTGCGGATACGAGAG) and TAP2R (5'-CGCAGTTCAGAATCAGCACC) was designed from published TAP2 sequence (29) and detects a 523-bp TAP2 product; ACT1 (5'-ACCTGACAGACTACCTCATGAA) and ACT23 (5'-ACTTGCGGTGCACGATGGAGG) detects a 570-bp -actin product (17). All primers were shown to be specific for the analyzed genes. The mixture was placed in a GeneAmp PCR System 9700 thermal cycler (PerkinElmer, Foster City, CA) and heated to 94°C for 15 min followed by 35 cycles at the settings of 94°C for 1 min for denaturation, 55°C for 1 min for annealing, and 72°C for 1 min for extension, followed by a final incubation at 72°C for 7 min. The PCR products were analyzed on a 1% agarose gel stained with ethidium bromide.

Generation of LAK cells

LAK cells were generated using a protocol adapted from previously published methods (2, 30, 31). Briefly, B6 spleen cells were aseptically harvested and cultured in complete DMEM medium supplemented with 500 U/ml murine rIL-2 (provided by Dr. M. Bennett). Cultures were maintained in 24-well plates in a total volume of 2 ml in a humidified incubator at 37°C 10% CO₂ and were harvested on day 5 or 6, unless indicated otherwise. LAK cells to be sorted were taken after 5 days of culture and were washed and resuspended in sterile sorting buffer (PBS with 1% BSA) at a concentration of 20 x 10⁶ cells/ml. A total of 4 x 10⁶ cells in a volume of 50 µl of sorting buffer were aliquoted into wells of a 96-well U-bottom plate. Five micrograms of Fc block was added to block FcR of cells to be stained and cells were incubated for 20 min on ice. Cells were then incubated for 30 min on ice with 40 µg/ml FITC-conjugated anti-TCR -chain mAb and 20 µg/ml PE-conjugated anti-NK1.1 mAb in a total volume of 100 µl. Live cells of lymphocyte size by forward and side scatter were gated and then sorted by the FACStar^Plus (BD Biosciences). Collected cells were resuspended in complete DMEM with 500 U/ml IL-2 at a concentration of 2 x 10⁶ cells/ml. Depending on the number of cells recovered, cells were incubated in either 24-well flat-bottom or six-well flat-bottom plates. All cells were incubated overnight at 37°C 10% CO₂.

Generation of poly(I:C)-activated killer cells

Poly(I:C) activation was performed essentially as described by Chang et al. (8). Mice were injected i.p. with 200 µg of poly(I:C) (Sigma-Aldrich). After 18–20 h, spleen cells were harvested and cultured on tissue culture-treated plates for 1 h at 37°C. The nonadherent cells were used as effector cells.

Generation of Con A lymphoblasts

Con A-activated T cell blasts were generated by culturing 30 x 10⁶ freshly isolated spleen cells in 6 ml of complete DMEM supplemented with 3 µg/ml Con A type IV (Sigma-Aldrich) for 60–72 h. Cells were spun down and resuspended in HBSS. Viable cells were isolated via density gradient centrifugation by collecting the cells at the interface of HBSS and the lymphocytic-specific gradient isolymph (Gallard-Schlesinger, Carle Place, NY). Excess isolymph was removed by washing cells with 10 ml of HBSS.

Cytotoxicity assays

⁵¹Cr release assays were performed according to the protocol of Dr. M. Bennett’s laboratory (2), with few modifications. Briefly, effector cells at various E:T ratios in a volume of 100 µl were added to the wells of a U-bottom 96-well plate. Target cells were labeled by incubating 2 x 10⁶ cells in a volume of 200 µl with 150–200 µCi of Na⁵¹CrO₄ (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at 37°C and 5% CO₂. A total of 2000 ⁵¹Cr-labeled target cells in 100 µl of medium were added to each well, and the plates were incubated at 37°C and 10% CO₂ for 4 h. For anti-Qa-2 blocking experiments, target cells were preincubated with either 100 µg/ml F(ab')₂ 20-8-4 mAb or 100 µg/ml control IgG F(ab')₂ (Caltag Laboratories) for 15 min on ice, and Ab was present throughout the killing assay. For NK cell receptor blocking, effector cells were preincubated with 2.5–5 µl/well Ly-49R-specific F(ab')₂ for 15–30 min at 37°C before addition of target cells, and Ab was present throughout the killing assay. For removal of GPI-attached molecules from the surface of melanoma targets, cells were treated with 2 U of phospholipase C (PLC; Sigma-Aldrich) and 40 µg of brefeldin A (Sigma-Aldrich) during the 1-h ⁵¹Cr labeling incubation. A total of 0.4 µg of brefeldin A was then added to each well during the 4-h incubation with effector cells. After incubation for 4 h, 100 µl of the supernatant was removed from each well and transferred to Skatron macrowell tubes (Skatron, Sterling, VA). Radioactivity was counted in a Micromedic Gamma Counter (ICN Biomedicals, Costa Mesa, CA). Data are expressed as the percentage of specific release, calculated as follows: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. Maximum release was determined by incubating target cells with 100 µl of 1% SDS. All experiments were performed in triplicate.

	Results

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Melanoma targets expressing Q9, H-2K^b, and H-2D^b Ags

Although the Q9 and the classical H-2K and H-2D Ags are remarkably similar in their structural composition, they differ in specific details of Ag presentation pathways. For example, at low temperatures the bulk of the class Ia H chains reach the cell surface of TAP2-negative RMA-S cells, where they can be selectively stabilized by exogenously added class Ia-specific peptides (32). In contrast, <5–10% of Q9 H chains come out to the surface of TAP-negative cells (Ref. 33 and data not shown). Because the vast majority of peptide-receptive Q9 chains remain intracellular, it is not possible to efficiently stabilize Q9 on the surface of transfected RMA-S or other TAP-negative cells. This property precludes the use of the standard stabilization approach (34, 35, 36) for studies of Q9 in NK target recognition.

To overcome this difficulty, we sought to identify an NK-sensitive cell line that will support expression of Q9 in the absence of other class I Ags. Based on a number of different criteria, the B78H1 derivative (22) of B16 melanoma was selected as a recipient for Q9 transfections. Its most important properties can be summarized as follows (E. Y. Chiang and I. Stroynowski, manuscript in preparation): 1) B78H1 is deficient in H-2K^b and H-2D^b transcription (23) and stains negative for surface ₂m; 2) this phenotype is not altered by incubation with IFN-; 3) ₂m is constitutively transcribed in B78H1, suggesting that ₂m expression is intact and available for interactions with transfected class I H chains; 4) B78H1 is TAP2 negative, but this deficiency is reversed upon IFN- stimulation.

We generated a panel of B78H1 transfectants expressing Q9 or empty vector and characterized their phenotype with anti-Q9 and anti-₂m Abs (Fig. 1). The analysis of these transfectants showed that B78H1 expressing Q9 cDNA driven by a CMV promoter (Q9.A7 clone) expressed low but detectable cell surface levels of Q9-specific 1-1-2 and 20-8-4 epitopes, as well as ₂m-specific S19.8 epitope (Fig. 1A, Q9.A7 panel). None of these epitopes was detected on empty vector-transfected B78H1 (Fig. 1A, vector panel). Supertransfection of the Q9.A7 clone with a plasmid expressing TAP2 cDNA (Q9TAP.C1 clone) resulted in a detectable enhancement of cell surface Q9/₂m epitopes (Fig. 1A, Q9TAP.C1 panel). Independently generated Q9TAP.11 and Q9TAP.17 clones (Fig. 1C) recapitulated this phenotype but presented a more uniform display of Q9 proteins. The up-regulation of Q9 in TAP-positive cells is consistent with the predicted enhancement of the transport of peptide-filled Q9 Ags to the cell surface in TAP-positive cells compared with TAP-negative cells (33). Treatment of B78H1 transfectants with IFN- (Fig. 1A, right panels) also resulted in increases of surface Q9/₂m complexes, because this cytokine induced TAP2 transcription in transfected cells (Fig. 1B) and may have had additional stimulatory effects on the Q9 Ag presentation pathway. IFN- stimulation did not induce ₂m or class I expression on vector-transfected B78H1 (Fig. 1A) despite detectable up-regulation of TAP2 revealed by RT-PCR (Fig. 1B). The latter result formally demonstrates that the parental B78H1 tumor remains class I negative in IFN--treated cells and that this phenotype is independent of TAP2 expression status.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Expression of Q9, Kb, Db, and TAP in transfected B78H1 tumors. A, Flow cytometry analysis of untreated (left panels) and IFN--treated (right panels) B78H1 transfectants. Each transfectant was stained for expression of Q9 with mAb 1-1-2 (dark gray filled histograms), Kb with mAb Y3 (solid line), Q9/Kb with mAb 20-8-4 (dashed line), and 2m with mAb S19.8 (dotted line). Background staining with secondary Ab alone is shown as light gray profiles. B, RT-PCR analysis of TAP2 expression by B78H1 transfectants. Cells were cultured in the absence or presence of IFN-, as indicated. In unstimulated cells, TAP2 transcription is detected only in cells transfected with TAP2 construct (Q9TAP.C1). TAP2 expression is IFN- inducible in all transfectants. -actin was included as a control for cDNA integrity. C, Flow cytometry analysis of additional transfectants used in this study. Independently generated Q9+TAP2+ B78H1 transfectants Q9TAP.11 and Q9TAP.17 were stained with mAb 1-1-2 (thick solid line) or mAb 20-8-4 (dashed line). Kb+TAP2+ B78H1 transfectants KbTAP.1-2.9 and KbTAP.1-2.25 were stained with mAb Y3 (thick solid line) or mAb 20-8-4 (dashed line). Db+TAP2+ B78H1 transfectants DbTAP.1-3.4 and DbTAP.1-3.5 were stained with mAb 28-14-8. Thin lines in histograms represent staining with secondary Ab FITC-goat anti-mouse IgG alone.

Q9 expression inhibits cytotoxic activity of LAK cell populations in vitro

Many different methods are currently used to generate LAK cells with cytotoxic activity. We have initially chosen a classical approach used by Bennett et al. (2) as well as others (30, 31). B6 LAK cells prepared according to the protocols, whereby splenic cells were cultured with IL-2 for 5–6 days, typically contained a very large proportion (30–65%) of NK1.1⁺TCR⁺ LAK cells and a smaller fraction (<10%) of classical NK1.1⁺TCR^- NK cells (Fig. 2A). Four-color flow cytometry analysis of these LAK cells revealed that the NK1.1⁺TCR⁺ LAK cell population was composed primarily of CD8⁺CD4^- cells (Fig. 2B) and expressed predominantly CD8 heterodimers (Fig. 2C). NK1.1^-TCR⁺ T cells in LAK cell cultures were predominantly CD8⁺ or CD4⁺ T cells (Fig. 2B); NK1.1⁺TCR^- NK cells were CD4 or CD8 negative (data not shown). The LAK cell phenotype observed in our experiments is consistent with previously published descriptions (30, 31).

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Phenotype analysis of IL-2-generated LAK cells. B6 splenocytes cultured with IL-2 for 5–6 days were stained with mAb specific for TCR -chain (or CD3; data not shown), NK1.1, CD4, CD8, and CD8.2. A, TCR vs NK1.1 staining on lymphoid-gated live cells. Numbers indicate percentages of cells in the corresponding quadrant. Cells were then gated on the upper left quadrant (NK1.1-TCR+) or upper right quadrant (NK1.1+TCR+) for further cell surface marker analysis. CD8 vs CD4 (B) and CD8 vs CD8.2 (C) expression profiles of NK1.1-TCR+ and NK1.1+TCR+ cells. Percentages of cells within each quadrant are shown for each dot plot. The profiles are from a single experiment selected from four similar experiments. LAK cells stained with anti-CD3 mAb instead of anti-TCR -chain had nearly identical staining profiles.

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Q9 partially inhibits the cytotoxic activity of LAK cells against B78H1 targets. Day 5–6 IL-2-cultured LAK cells were used in 4-h 51Cr release assays against tumor targets. A, Lysis (as percentage of specific release) of vector-transfected and Q9/TAP2-transfected (Q9TAP.C1) targets as a function of E:T ratio. Inset, Sensitivity of vector-transfected B78H1 () to LAK-mediated killing relative to YAC-1 () and P815 () control targets. Results are shown as mean ± SE. One representative experiment of over 25 performed is shown. B, Q9 inhibition of LAK cytotoxic activity is statistically significant. The ability of IL-2-generated LAK cells to lyse three independently generated Q9-expressing targets was examined. Lysis of Q9/TAP2 transfectants was compared with that of vector-transfected B78H1 at two E:T ratios: 100:1 and 30:1. Percentage of inhibition of lysis was calculated as [1 - (Q9TAP % specific lysis/vector % specific lysis)] x 100. Percentage of inhibition of lysis for Q9TAP.C1 was 31.2 ± 2.2% at 100:1 and 30.8 ± 3.4% at 30:1; percentage of inhibition of lysis for Q9TAP.11 was 26 ± 2% at 100:1 and 28 ± 4.3% at 30:1; percentage of inhibition of lysis for Q9TAP.17 was 29.9 ± 3.2% at 100:1 and 34.7 ± 5.9% at 30:1. Inhibition was significant in all cases by Student’s t test (*, p < 0.0005). Data are expressed as mean ± SE.

Ab against Q9 restores LAK cell-mediated lysis of Q9-positive targets

The differential cytotoxic activity of bulk LAK cells against B78H1 tumor and Q9 transfectants suggested that cell surface display of Q9 Ag partially inhibited the lysis of the Q9-positive targets. To confirm this interpretation, we tested whether Abs blocking the access to Q9 during the killing assay would interfere with the inhibition. F(ab')₂ of 20-8-4 mAb, which interacts with ₁₂ domains of Q9 (37), were used to diminish the possibility of Ab-dependent cellular cytotoxicity. At the concentration of 100 µg/ml, the anti-Q9 blocking F(ab')₂ fully restored killing of the Q9TAP.C1 targets but had no effect on killing of the vector-transfected tumors (Fig. 4A). As an additional control, we tested F(ab')₂ of an isotype-matched control Ab. At the same 100 µg/ml concentration, the control F(ab')₂ neither blocked nor enhanced the killing of vector-transfected B78H1 or Q9TAP.C1. Titration of the blocking anti-Q9 F(ab')₂ demonstrated that full restoration of killing is observed at concentrations greater than 10 µg/ml (Fig. 4B). Similar data were observed with two other Q9-expressing targets (data not shown). These results indicate that cell surface expression of Q9 is likely to be responsible for inhibiting LAK cell cytotoxicity in the in vitro assays.

View larger version (69K): [in this window] [in a new window]   FIGURE 4. Antibody against Q9 completely restores LAK cell cytotoxic activity against Q9-expressing B78H1 targets. A, Vector-transfected B78H1 or Q9TAP.C1 targets were preincubated with either no blocking Ab, 100 µg/ml anti-Q9 blocking 20-8-4 F(ab')2 Ab, or 100 µg/ml control F(ab')2 Ab, then incubated with day 6 LAK cells at an E:T ratio of 100:1 for 4 h at 37°C. This experiment was performed six times, with the results shown above. B, Target cells were preincubated with increasing concentrations of anti-Q9 blocking 20-8-4 F(ab')2 Ab, then incubated with day 6 LAK cells at an E:T ratio of 100:1 for 4 h at 37°C. In data not shown, preincubation with control F(ab')2 Ab had no effect on susceptibility of targets to effector cell cytolysis. Killing in the presence of control F(ab')2 Ab was similar to killing without Ab at all concentrations. The blocking Ab titration experiment was performed twice with similar results. All Ab blocking treatments were conducted in triplicate, with data shown as mean ± SE.

Q9 Ags are attached to the cell membrane via GPI lipid anchors that are sensitive to cleavage by PLC (38). To further demonstrate that inhibition of killing is regulated by Q9 expressed on target cells, B78H1 target cells were treated with PLC before incubation with LAK cell effectors to remove Q9 from the cell surface. In addition, brefeldin A was added to block egress of newly synthesized Q9 Ags to the cell surface after PLC cleavage. This combined treatment resulted in complete elimination of Q9 from the surface of Q9-positive B78H1 transfectants throughout the culture conditions simulating the cytotoxicity assay (Fig. 5A). The PLC-treated and untreated Q9 transfectants exhibited comparable levels of killing by LAK cells (Fig. 5B). In contrast, survival of vector-transfected tumor cells was enhanced by 30–45% compared with conditions that did not include PLC and brefeldin A (Fig. 5C). This pattern is most consistent with an explanation that PLC/brefeldin A reduced LAK cytotoxicity toward both targets, but removal of Q9 from Q9 transfectants compensated reproducibly for this effect by enhancing Q9 but not vector target sensitivity. The differential effect of PLC/brefeldin A on killing of targets lends further support for the protective effect of Q9 expression in LAK cell-mediated killing.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Removal of GPI-linked surface molecules affects the ability of LAK cells to recognize and kill B78H1 tumor cells. A, Histogram profiles of Q9-expressing Q9TAP. C1 with (right panel) or without (left panel) PLC treatment. Thick line, Qa-2 surface expression as detected using 1-1-2 mAb; thin line, background staining with irrelevant control Ab. Cells were stained after conditions simulating 4-h cytotoxicity assays. Cells were incubated with PLC and brefeldin A for 1 h, washed thoroughly, then incubated for 4 h in the presence of brefeldin A as described in Materials and Methods. B and C, In vitro cytotoxicity assays using untreated or PLC/brefeldin A-treated Q9TAP. C1 cells (B) or vector-transfected B78H1 cells (C) as targets and day 5 LAK cells as effectors. Triplicate determinations were made at each E:T ratio, and results are shown as mean ± SE. Data shown are representative of four experiments.

Regulatory effects of class Ia Ags displayed on B78H1 targets

Our results suggest that Q9 has a modest, 30% down-regulatory effect on bulk LAK cell killing. It is important to emphasize that the degree by which Q9 inhibits LAK cells in this system is, by itself, insufficient to draw conclusions about the strength of the signal induced by LAK cell/Q9 interactions or the clonal distribution/density of the putative Q9-reactive LAK cell inhibitory receptors (see Discussion). To provide an independent reference for the B78H1 killing/inhibitory effects, we tested whether H-2K^b, a known NK regulatory element that can inhibit selected NK populations (2, 34, 36), has a detectable influence on killing of B78H1 tumor. Using B78H1 cells cotransfected with K^b and TAP2 (K^bTAP.1-2.9 and K^bTAP.1-2.25 in Fig. 1C) as targets, we showed that the inhibitory effects of K^b were very similar to those of Q9 (Fig. 6A, 35% inhibition at 100:1 E:T ratio) and quantitatively comparable to inhibitory effects of peptide-filled K^b expressed on RMA-S or blasts (2, 34, 39). In contrast, B78H1 targets expressing TAP2 and H-2D^b (D^bTAP.1-3.4 and D^bTAP.1-3.5) were not protected from killing by LAK cells (Fig. 6B). This result is consistent with previous findings in which D^b-positive hemopoietic cells were used as targets (2, 39).

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Expression of class Ia MHC H-2Kb, but not H-2Db, protects B78H1 tumor cells from LAK cell-mediated cytolysis. The ability of IL-2-generated LAK cells to lyse two independently generated Kb+TAP2+ clones (A, KbTAP.1-2.9 () and KbTAP.1-2.25 ()) and Db+TAP2+ clones (B, DbTAP.1-3.4 () and DbTAP.1-3.5 ()) was examined. One representative experiment of four is shown. Data are shown as mean ± SE. Percentage of inhibition of lysis was calculated as described in Fig. 3 by comparison with lysis of vector-transfected B78H1 (). Percentage of inhibition of lysis at 100:1 E:T ratio for Kb+TAP2+ clones ranged from 27 to 56%. No significant inhibition was provided by Db expressed on B78H1 targets (ranging from 0 to 11%).

To determine whether inhibition of LAK cells by Q9 molecules is dependent on a threshold level of surface expression on target cells, we compared the killing of TAP-negative Q9^low and TAP2-positive, IFN--treated, Q9^high Q9.A7 transfectants (characterized in Fig. 1A). The results in Fig. 7 demonstrate that the constitutively expressed, heat-stable Q9 on Q9.A7 did not reduce LAK cell cytotoxicity to a statistically significant degree. In contrast, TAP2-enhanced levels of Q9 present on IFN--treated Q9.A7 cells were sufficient to down-regulate cytotoxicity by 30–50%. These results are consistent with the density-dependent effect of Q9. However, we cannot exclude a possibility that the nature of the peptides/ligands bound to Q9 under the TAP2-negative vs TAP2-positive (and IFN--induced) conditions contributes to these observations. For instance, specific sets of peptide/Q9 complexes that can differentially influence LAK cell activity may be favored.

View larger version (43K): [in this window] [in a new window]   FIGURE 7. Q9 regulation of LAK cell cytotoxicity is dependent on surface expression levels. Vector- and Q9 (Q9.A7)-transfected B78H1 tumor cells were cultured in the absence or presence of IFN- for 3 days before use as targets in 4-h 51Cr release assays. Labeled target cells were incubated with day 6 LAK cells at E:T ratios of 100:1 or 30:1. Lysis of Q9.A7 was compared with vector for the same culture condition and percentage of inhibition was calculated as in Fig. 3B. In the absence of IFN-, Q9 is expressed at low levels (Fig. 1A) and no statistically significant inhibition is observed (n = 4; percentage of inhibition = 11.9 ± 6% at 100:1, p > 0.05; percentage of inhibition = 2.8 ± 15.5% at 30:1, p > 0.10). Q9.A7 cells stimulated with IFN- express relatively high levels of surface Q9 (Fig. 1A) and are significantly resistant to killing (n = 4; percentage of inhibition = 30.9 ± 2.1% at 100:1 and 43.4 ± 0.9% at 30:1, n = 4; * p < 0.0005 by Student’s t test).

Because Q9 is structurally invariant and its polymorphism affects predominantly quantitative expression, we asked whether the putative Q9-reactive LAK cell inhibitory receptors are functionally present in different strains. To address this point, we examined the killing of Q9 and control tumor targets by LAK cells isolated from Qa-2-negative B6.K1 (H2^b, Qa-2^null) and C3H (H2^k, Qa-2^null) and Qa-2-positive 129 (H2^b, Qa-2^high) and BALB/c (H2^d, Qa-2^med) inbred mice. The results in Fig. 8 demonstrate that LAK cells from these different mouse strains were sensitive to down-regulation of LAK cell activity by Q9. The quantitative variations discerned between the different strains were not statistically significant and we do not know, at present, whether they represent true differences. The results are most consistent with the notion that the Q9 inhibitory effect on LAK cells is achieved via a conserved receptor and that presence of this putative receptor in mice is not detectably affected by the presence/absence of Q9 or differences in H2 haplotypes.

View larger version (40K): [in this window] [in a new window]   FIGURE 8. Q9 inhibits activity of LAK cells derived from both Q9+ and Q9- mouse strains of different haplotypes. Day 5–6 LAK cells were generated from Qa-2-negative B6.K1 (H2b) and C3H (H2k) and Qa-2-positive 129 (H2b) and BALB/c (H2d) mice in the same manner as B6 LAK cells. All effectors were tested against vector-transfected B78H1 and at least one Q9-expressing transfectant. Results are shown as mean ± SE. Experiments were performed at least three times with effector cells from each mouse strain.

Different populations of NK1.1⁺ cytotoxic LAK cells are inhibited by Q9

Our experiments with IL-2-cultured LAK cells did not identify the active populations of cytotoxic cells that can be inhibited by Q9. Based on previous studies (30), we reasoned that the two most likely candidate effectors are classical NK1.1⁺TCR^- NK cells, which represent a minor fraction (<10%) of our LAK cells, and NK1.1⁺TCR⁺ LAK cells, which represent a major fraction of IL-2-derived LAK cells (30–65%). To directly address whether Q9 inhibits activity of NK cells, we used effectors from SCID mice, which do not produce mature T cells or B cells and lack CTL as well as IL-2-induced NK1.1⁺TCR⁺ LAK cells. The flow analysis in Fig. 9A confirms this point and identifies NK cells as the major population of SCID LAK cells. The results in Fig. 9B indicate that the NK1.1⁺ cells from SCID killed B78H1 and that this killing was reduced in the presence of Q9. Furthermore, inhibition of killing was reversed in the presence of anti-Q9 F(ab')₂ (Fig. 9C). This demonstrates that NK cell-mediated cytotoxicity against B78H1 can be inhibited by Q9 Ag.

View larger version (43K): [in this window] [in a new window]   FIGURE 9. Q9 inhibits "classical" NK cells. Effector cells were generated by culturing splenocytes from SCID mice in the presence of IL-2 for 5–6 days. A, Multicolor flow cytometry analysis of effector populations. SCID LAK cells were stained for TCR and NK1.1. Percentages of cells for each quadrant are shown below the dot plot. In data not shown, cells were gated on the TCR-NK1.1+ (lower right) quadrant and analyzed for CD8 vs CD4 and CD8 vs CD8.2 expression. These cells were 95% negative for these surface markers. Profile shown is representative of three separate stainings. B, SCID LAK cells were used against vector-transfected and Q9-expressing Q9TAP.C1 B78H1 targets. Inset, Sensitivity of vector-transfected B78H1 () to LAK-mediated killing relative to YAC-1 () and P815 () control targets. Results are shown as mean ± SE. One representative experiment of five is shown. C, Ab against Q9 completely restores NK cell-mediated cytotoxicity against Q9-expressing targets. SCID LAK cells were tested against target cells preincubated with F(ab')2 Ab as described in Fig. 4. Percentage of specific lysis was determined at an E:T ratio of 100:1, and data are shown as mean ± SE.

View larger version (42K): [in this window] [in a new window]   FIGURE 10. NK1.1+TCR+ LAK cells are inhibited by Q9. A, Cell sorting of NK1.1+TCR+ LAK cells. Day 5 bulk LAK cells were stained with mAb specific for TCR -chain and NK1.1. Gate (R2) was set on cells that were double positive for NK1.1 and TCR, yielding a highly purified NK1.1+TCR+ postsort population of LAK cells. B, Sorted NK1.1+TCR+ cells were cultured overnight and then used as effectors in 4-h 51Cr release assays. For comparison, unsorted bulk LAK cells from which the sorted population was obtained were also tested. Triplicate measurements were made at an E:T ratio of 50:1 and data are shown as mean ± SE. This experiment was performed twice with nearly identical results. C, NK1.1+TCR+ LAK cells express Ly49 inhibitory receptors. Day 5 bulk LAK cells were triple stained with mAb specific for TCR -chain, NK1.1, and Ly49C/I (using 5E6 mAb). The histogram profile shows 5E6 expression on NK1.1+TCR+-gated subset of cells.

Up to this point, all our experiments used IL-2-activated LAK cells as effectors. Because this procedure generated a biased composition of NK1.1⁺TCR⁺ LAK cells over classical NK cells, we prepared activated NK cells by in vivo injection of poly(I:C). Comparison of TCR/NK1.1 expression on splenocytes from poly(I:C)-injected mice (Fig. 11A) with that from uninjected mice (Fig. 11B) showed that the experimental and the control animals had similar proportions of NK1.1⁺TCR⁺ and NK1.1⁺TCR^- cells. Both the poly(I:C)-activated as well as the nonactivated splenocytes killed YAC-1 targets, albeit with lower efficiency than LAK cells. In contrast, vector-transfected B78H1 targets were detectably killed only by poly(I:C)-activated cells. As observed with LAK cells, the poly(I:C)-induced killing was reduced by expression of Q9 on target cells (Fig. 11A). These results suggest that Q9 offers protection from poly(I:C)-induced cytotoxicity against B78H1. The differential killing of B78H1 and YAC-1 tumors by poly(I:C) activated vs nonactivated splenocytes indicates that the B78H1-specific effectors may depend on activation through a different pathway than YAC-1 killers. Alternatively, the threshold for killing of YAC-1 by constitutively activated NK cells is lower than the threshold for killing of B78H1. Furthermore, absence of detectable B78H1 killing by normal splenocytes provides formal evidence that poly(I:C) nonresponsive splenic populations do not contribute to the cytotoxicity against B78H1 in our in vitro assays.

View larger version (49K): [in this window] [in a new window]   FIGURE 11. Q9 inhibits the ability of poly(I:C)-activated NK cells to lyse B78H1. Spleen cells harvested from mice injected i.p. with poly(I:C) (A) or freshly isolated splenocytes from untreated mice (B) were used as effectors against 51Cr-labeled vector and Q9TAP.11 B78H1 transfectants. Cells were stained with mAb specific for TCR and NK1.1. Numbers indicate percentages of cells in each corresponding quadrant. Data points in killing assays are mean ± SE. Similar results were obtained in four separate experiments.

This study demonstrates that Q9 expressed on the surface of neural crest-derived melanoma cells inhibits NK and LAK cells. To test whether Q9 expressed on nontransformed/other tissue type cells has the same protective effect, LAK cells were tested in in vitro cytotoxicity assays against Con A splenic blast targets derived from different B6-background mouse strains. The panel included Q9-positive strains B6 and K^b-/-D^b-/- (deficient for class Ia, expressing class Ib; Ref. 14) and Q9-negative strains B6.K1 (lacking Qa-2 genes; Ref. 44), and ₂m^-/- and TAP^-/- (deficient for class Ia and many class Ib, including Qa-2; Refs. 39 and 45, 46, 47, 48). Flow cytometry studies in Fig. 12A establish that T cell blast surface levels of K^b, Qa-2, and ₂m are reduced at least 10-fold in ₂m^-/- or TAP^-/- mice compared with B6, while T cell blasts of K^b-/-D^b-/- mice retain 30% of their surface ₂m. The great majority of class Ib molecules associated with ₂m in K^b-/-D^b-/- are likely to be Qa-2, as the Qa-2 expression levels on T cells approach H-2K (49). Nevertheless, we find that K^b-/-D^b-/- blasts are as sensitive to LAK cell killing as TAP^-/- or ₂m^-/- blasts (Fig. 12B). The class Ia-expressing blasts, B6 and B6.K1, block LAK cell cytotoxicity to a degree indistinguishable from each other under the conditions of our experiments. These results confirm earlier observations (39, 45, 46, 47, 48) and suggest that Q9 regulatory effects on NK cells may depend on the target cell on which Q9 is expressed.

View larger version (33K): [in this window] [in a new window]   FIGURE 12. Killing of B6, 2m-/-, TAP-/-, and Kb-Db- Con A blasts by IL-2-activated LAK cells. A, Flow cytometry histograms of blasts derived from various mouse strains, as indicated for each profile. Blasts were stained for Qa-2 (bio-M46, upper histograms), Kb (bio-Y3, middle histograms), and 2m (bio-S19.8, lower histograms). The degree of biotinylation for each Ab is variable and the specific activity of bio-M46 is lower than that of bio-Y3. The light gray shaded area is irrelevant control Ab staining. B, 51Cr-labeled Con A-activated T cell blast target cells were incubated with day 5–6 B6 LAK effector cells for 4 h. Results are shown as mean ± SE. One representative experiment of three is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In B6 mice, only Ly49C/I of the known Ly49 inhibitory receptors has a clearly defined specificity for self (H2^b) class Ia Ags (8). In addition, the CD94/NKG2A recognizes self H-2D^b leader peptide in the context of Qa-1. Importantly, staining of NK cells with Abs and/or tetramers against these two classes of receptors (5, 6, 52) and single cell RT-PCR analyses (53) revealed that approximately one-fourth of NK cells in B6 mice do not express either of these known receptors to self (8). Furthermore, extensive polymorphisms of MHC class Ia ligands and their independently inherited Ly49 receptors suggest that NK cells of individuals who inherit selected sets of Ly49 receptors/MHC alleles may fail to recognize their own class Ia Ags. Similarly, because CD94/NKG2A binds only a subset of the known class Ia leader peptides, the inhibition mediated through this receptor may be inactive in some haplotypes. This may also occur during transcriptional shutdown of class Ia genes in certain virally infected or malignantly transformed cells or in immunologically privileged sites where expression of class Ia is very low. The potential self-reactivity of NK cells in syngeneic systems raises a question of whether additional inhibitory receptors/class Ia ligands contribute to the maintenance of tolerance to self cells.

We report in this work that the nonpolymorphic Q9 Ag can inhibit cytolytic activity of NK cells and LAK cells, and we propose that this phenomenon may be due to the interaction of Q9 with conserved inhibitory receptor(s) expressed on NK and other activated cells. The Q9 Ag is one of the best-characterized murine class Ib molecules, yet its physiological function(s) remains unknown. This MHC molecule shares many similarities with class Ia proteins: it binds ₂m and a wide repertoire of peptides delivered by TAP-dependent pathway (33); its tissue distribution includes hemopoietic and nonhemopoietic cell types; and the pattern of expression, while quantitatively different, overlaps with H-2K, H-2D, and H-2L (21). It also has some unique properties: it is attached to cell surfaces via GPI (38) and, upon cross-linking with Abs, activates T cells on which it resides (54, 55). Like many other class Ib molecules, such as HLA-G (56), Q9 are also synthesized as soluble molecules (27, 38). The diverse soluble forms of Q9 are found in murine blood and their levels are quantitatively induced by poly(I:C) with kinetics coinciding with induction of NK activation (57). Although Q9 is present on thymic epithelial cells (14, 21), it fails to select substantial populations of CD8⁺ T cells (14, 15), thus suggesting that it is unlikely to be an efficient restriction element for pathogen-restricted T cells (14, 15). Despite this possible deficiency in T cell immunity, Q9 binds endogenous as well as viral/bacterial peptides (12), raising a possibility that it may function independently of class Ia in recognition of self vs nonself. Consistent with this assumption is the observation that the transcription of Q9 is modulated independently of class Ia in many tumors (21).

To test whether Q9 interacts with NK cells, we used the B16 melanoma-derived B78H1 tumor and its transfected derivatives. The salient features of the B78H1 melanoma include the total absence of all ₂m-dependent class I Ags on the cell surface of the parental tumor and its high susceptibility to LAK cell-mediated killing. In the present study, we demonstrated that display of Q9 on the B78H1 surface led to reduced killing of the targets by splenic IL-2-induced LAK cells and that this inhibitory effect was specifically prevented by anti-Q9 F(ab')₂ Ab fragments. Further evidence supporting the protective role of Q9 was provided by PLC treatment of Q9-expressing targets. Enzymatic removal of Q9 from the cell surface resulted in cytolytic activity that compensated for the reduction in overall capacity of LAK cells to recognize and kill the tumor targets.

We have also examined the nature of the effector cells responsible for killing of the B78H1 targets and showed that different activation protocols such as poly(I:C) injection in vivo or culturing of splenic cells with IL-2 in vitro lead to generation of cytolytic cells inhibitable by Q9. Experiments with LAK cells derived from SCID mice established that NK cells were the active cytolytic population that is inhibited by Q9 in this strain. In B6 mice, the most prevalent lytic effectors generated by IL-2 activation were NK1.1⁺TCR⁺CD8⁺ LAK cells. These CD1d-independent splenic NK1.1⁺TCR⁺ cells, characterized previously by other laboratories (30, 31), are thought to arise from NK1.1^-CD8⁺IL-2R⁺ precursor T cells upon IL-2 incubation in vitro or viral infection in vivo (31) and are known to express a variety of NK cell receptors (31). We showed in our study that these CD8⁺ LAK cells are inhibited by Q9 to a degree comparable to that seen with NK cells from SCID mice. Taken together, the results are most easily reconciled with the hypothesis that the nonpolymorphic Q9 protects the syngeneic tumor cells from LAK cell cytotoxicity by engaging conserved inhibitory receptors expressed on activated NK1.1⁺ cells.

The nature of this putative receptor is speculative at this time. It is possible that NK cell receptors recognizing Q9 correspond to one or more of the known murine inhibitory receptors from the Ly49, CD94/NK2G, or paired Ig-like receptor PIR (58)-related families, or perhaps different, as yet unidentified, proteins. To date, 21 Ly49 proteins have been detected based on reactivity patterns of mAbs. Of these, Ly49A, B, C, E, F, G, I, J, O, Q, S, T, and V have been shown or are predicted to be inhibitory receptors (40, 41). However, not all mouse strains express all of these receptors, and of those receptors that are common to several strains, polymorphisms may exist (43). LAK cells from different strains were inhibited by Q9 to a similar degree, suggesting that the protective effect of Q9 is not detectably influenced by Ly49 polymorphism among the strains (B6 vs 129 vs BALB/c vs C3H) or the presence or absence of Q9 in the NK harboring hosts (B6 vs B6.K1 vs C3H). Furthermore, Ab blocking of Ly49C/I and G2, the Ly49 inhibitory receptors that are expressed by the four strains used in this study, did not restore killing, suggesting that Q9 is not a ligand for any of these known inhibitory receptors. The possibility exists that Q9 may interact with numerous different inhibitory receptors, and that blocking of one or two members is insufficient to remove the inhibitory effect. For example, the class Ia MHC molecule H-2D^d is a putative ligand for inhibitory receptors Ly49A, C, and G expressed in B6 mice (40). Experiments using a mixture of 5E6 and 4D11 have been inconclusive (data not shown).

Because H-2K^b expressed on B78H1 TAP-positive targets reduced LAK cell killing in our in vitro assays to approximately the same degree as Q9, one could propose that the Q9-specific inhibitory receptor is expressed on a similar proportion of LAK cells as K^b-reactive Ly49C/I (2), which we detected on 12–25% of NK cells (data not shown) and 20–30% of NK1.1⁺TCR⁺ LAK cells (Fig. 9C) in bulk LAK cell cultures. An important caveat of such a proposition is that the observed results may be influenced by qualitative differences in the putative receptor interactions with their respective class I ligands. The class Ia-mediated NK cell inhibition has been previously reported to depend on the density of K^b ligands on target cells (59). In this study we also presented evidence suggesting that the Q9-mediated NK inhibition may require a minimal threshold of Q9 on the surface of target cells. It is thus conceivable that the Q9-mediated inhibition is dependent on the density of Q9 on the target cells in a way that differs from K^b interactions with Ly49C/I. It is also possible that the negative signals delivered to NK cells by K^b and Q9 are qualitatively and/or quantitatively different in counterbalancing the activation pathway against B78H1 tumor. Finally, Q9 may reduce the killing of LAK cells by as yet unknown mechanisms that are not paralleled by Ly49C/I/H-2K^b signaling.

The inhibitory effect of Q9 on LAK cell-mediated cytotoxicity is clearly demonstrated when Q9 is expressed on B78H1 tumor cells. However, Q9 expression on T cell blasts does not seem to confer the same protection. B6 blasts expressing their full repertoire of class I molecules are recognized as self by B6 LAK cells and are thus spared from lysis. Q9 is not expressed by B6.K1 blasts, but these too are resistant to LAK cells because other class I molecules such as K^b are present. Targets lacking class I such as ₂m^-/- and TAP^-/- blasts are readily lysed by LAK cells. Interestingly, blasts expressing Qa-2 in the absence of class Ia molecules (K^b-/-D^b-/- blasts) are killed to the same degree as ₂m^-/- blasts. This finding recapitulates observations of Grigoriadou et al. (39), who also recently reported that inhibition of NK cells involved in bone marrow graft rejection requires negative signaling from H-2K^b and H-2D^b, but found no evidence that class Ib molecules contribute significantly to this process. Their conclusions were based on experiments in which B6 mice were engrafted with bone marrow from mutant K^b-/-, D^b-/-, or K^b-/-D^b-/- mice or experiments in which IL-15-activated LAK cells or poly(I:C)-activated NK cells were tested for killing of class Ia-deficient Con A blasts. Several differences between B78H1 tumor cells and hemopoietic cells may explain the inability of Qa-2 to protect blast targets from LAK cell-mediated cytolysis. B78H1 does not express ₂m-dependent class I Ags, while K^b-/-D^b-/- blasts express other class Ib molecules in addition to Qa-2. It is conceivable that the full complement of class Ib proteins expressed in K^b-/-D^b-/- mice influences negative as well as positive NK regulatory signals and that the net effect of these interactions is not detectable with blast targets. Alternatively, NK cells activated by blasts may require qualitatively different inhibitory signals than NK cells activated by tumors or nonhemopoietic cells. A precedent for different activation pathways being inhibited differentially by class I has been reported for RMA vs RAE-1-transfected RMA cells (60). It is also possible that peptides presented in the context of Qa-2 on T cell blasts vs melanomas may have an influence on the inhibitory effect, as peptide dependence has been observed with some human and murine NK cell receptor/class I interactions (34, 61). In this respect it is of interest that peptide resides higher up in the shallow groove of Q9 than peptides in other MHC molecules (11). Peptide presented in the context of Q9 also has a very high accessibility to solvent, suggesting that it is more likely to interact with other ligands than peptides buried deeper in MHC pockets (11).

The NK inhibitory effects of Q9 molecules are interesting in the context of the known biological properties of this class Ib Ag. Q9 are expressed as both membrane-bound and soluble proteins in several immunologically privileged sites where expression of class Ia is low and NK cell attack may be detrimental (17, 18, 19, 20, 21). These features are reminiscent of the human class Ib molecule HLA-G, the major inhibitory NK ligand recognized by killer cell inhibitory receptor (62), leukocyte Ig-like receptor (63), and CD94/NKG2A (64) receptors in placenta. Q9 also shares similarities with HLA-C, a ubiquitously expressed human class Ia Ag that functions as a dominant ligand for human killer cell inhibitory receptors (65) and is rather ineffective as a restriction element for T cells (66). The separation of NK cell and T cell functions in HLA-G and HLA-C (67) allows them to avoid "compromises" facing other class Ia molecules confronted with opposing selections by T cells and NK cells.

Recent evidence suggests that many molecules dominating mouse and human NK cell/class I recognition evolved independently in these species but nevertheless show functional homologies (68). Q9 and HLA-G/HLA-C may constitute another example of convergent evolution. It appears that NK cell-regulating properties of these molecules developed at the expense of their T cell-relevant functions. This specialized adaptation was convincingly demonstrated for HLA-G and HLA-C and allows them to confer selective advantage to cells threatened with destruction by NK cells (67). It remains to be established whether similar mechanisms operate on Q9, when it down-regulates cytotoxicity of NK cells.

	Acknowledgments

 
We are grateful to Drs. Michael Bennett, Thaddeus George, John Schatzle, Maria Johansson, and Deming Zhou for scientific discussions, advice, and valuable reagents; Kathleen Robertson, Jill Mooney, and Dr. Piotr Tabaczewski for their contributions in generating B78H1 transfectants; and Angie Mobley, Threedanuj Ungchusri, and Bonnie Darnell for expert assistance in flow cytometry and cell sorting.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants RO1 AI 19624, PO1 AI 37818, and T32 CA 09082.

² Address correspondence and reprint requests to Dr. Iwona Stroynowski, Center for Immunology, Departments of Microbiology and Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9093. E-mail address: stroynow{at}utsw.swmed.edu

³ Abbreviations used in this paper: LAK, lymphokine-activated killer; ₂m, ₂-microglobulin; PLC, phospholipase C; bio-, biotinylated.

Received for publication May 4, 2001. Accepted for publication December 20, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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